CN114080235A - Treatment of patients undergoing treatment with antiplatelet drugs that experience bleeding - Google Patents

Abstract

The present invention relates to platelet endothelial aggregation receptor 1(PEAR1) agonists for use in treating bleeding in a patient, wherein the patient is receiving antiplatelet drug therapy. Also provided are topical hemostatic, sealant or adhesive agents for topical use during surgery.

Description

Treatment of patients undergoing treatment with antiplatelet drugs that experience bleeding

Technical Field

The present invention relates generally to the treatment of patients under antiplatelet drug therapy with agents that modulate human platelet activation and aggregation. Particularly patients who experience bleeding complications during/after surgery. Furthermore, the present invention relates to platelet endothelial receptor 1(PEAR1) agonists such as natural fucoidan, synthetic fucose-mimicking glycopolymers such as sulfated alpha-L-fucoside chain glycopolymers or dextran sulfate for such use.

Background

Human platelets, also colloquially referred to as platelets, patrol blood vessels as sentinels of vascular integrity. Platelets play a key role in physiological hemostasis by adhering to damaged vessel walls, clotting, the propagation of blood coagulation, and thrombosis (the latter also known as "blood clots"). In addition, platelets are subsequently involved in fibrinolysis (breakdown of the thrombus) and repair of the vessel wall, restoring blood flow and vessel integrity.

In pathophysiological conditions such as atherosclerosis, rupture of atherosclerotic plaques can lead to inappropriate platelet aggregation and thrombosis, which can lead to vascular occlusion leading to Acute Myocardial Infarction (AMI) or stroke, which are major causes of death worldwide according to the World Health Organization (WHO).

Briefly, thrombus formation involves two main components: activated platelets and soluble blood components (the latter being called "coagulation factors"), and which form the "coagulation cascade". In addition to releasing autocrine feedback mediators such as thromboxane a2(TXA2) and Adenosine Diphosphate (ADP) and establishing loose fibrinogen-bridged aggregates, activated platelets also change the composition and charge of their outer membrane to a so-called "procoagulant surface". Coagulation factors bind to the surface and convert to their active form, ultimately converting fibrinogen to fibrin, thereby converting loose platelet aggregates to stable thrombi.

In this regard, antithrombotic agents fall into two broad groups of agents. One group is anticoagulants, targeting components of the coagulation cascade, such as heparin or vitamin K antagonists. The other group is antiplatelet drugs, which directly interfere with the initial activation of platelets. Thus, anticoagulants allow platelet aggregation but block coagulation, while antiplatelet drugs block initial platelet activation and aggregation, and thus similarly block coagulation. Anticoagulants are used primarily in patients who exhibit clinical indications that may lead to future thrombotic events. In contrast, antiplatelet drugs are administered immediately at high doses to patients experiencing AMI or stroke. Patients who survive such an event must often receive lifelong treatment with antiplatelet drugs to prevent the recurrence of such life-threatening complications. Along this route, the combination of drugs that interfere with TXA2 synthesis and signaling, such as aspirin, with antagonists of the P2Y12 receptor of ADP, such as clopidogrel (clopidogrel), cangrelor (cangrelor) or ticagrelor (ticagrelor), represents the "gold standard" for dual antiplatelet therapy (DAPT) today to treat acute events and their recurrence. If such a patient requires a surgical or dental procedure that can be planned in advance, it is currently preferred to temporarily stop the medication in a controlled manner. However, bleeding complications are inevitable when patients under antiplatelet drug therapy urgently need immediate/acute surgery.

However, in rare cases, patients may be misdiagnosed as experiencing AMI or stroke because of the symptoms displayed and the relatively high incidence of such events, and will therefore be immediately administered an ultra-high dose of a fast-acting P2Y12 receptor antagonist, such as clopidogrel, cangrelor, or ticagrelor. Immediate surgery is inevitable when a patient is subsequently diagnosed as experiencing, for example, acute Aortic Dissection (AD) rather than AMI or stroke. The surgical method of choice is to divide the aorta and suture a surgically inserted graft. At the junction between the aorta and the graft, as well as at the suture, bleeding is prolonged due to the lack of platelet aggregation, clotting, and embolization, which is a direct result of the initial receipt of an ultra-high dose of P2Y12 receptor antagonist.

Currently, there is no effective hemostatic solution in the above situations. Existing products are topical hemostats, sealants and adhesives. Hemostatic agents can coagulate blood. The sealant can create a hermetic barrier. The adhesive bonds the tissues together. Collagen, gelatin and cellulose are hemostatic agents.

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development of H. The TachoComb is made from horse collagen, bovine thrombin, bovine aprotinin and human fibrinogen. However, none of these products effectively address the bleeding complications during surgery caused by P2Y12 receptor blockade or DAPT drugs.

The lack of methods or products to address complications caused by P2Y12 receptor antagonists or DAPT during acute surgery often results in patients who may have to endure an open chest wound and receive a large number of transfusions within days, both of which carry an additional serious risk of infection until bleeding stops and the surgeon can close the wound.

Therefore, there is a need for therapeutic strategies to address bleeding complications in patients under treatment with antiplatelet drugs, especially those patients who are urgently in need of immediate surgery

Disclosure of Invention

Accordingly, the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above mentioned problems by providing a platelet endothelial aggregation receptor 1(PEAR1) agonist for use in the treatment of bleeding in a patient, wherein the patient is under treatment with an antiplatelet drug.

Also provided are topical hemostatic, sealant or adhesive for topical administration during surgery, wherein the topical hemostatic, sealant or adhesive comprises a platelet endothelial aggregation receptor 1(PEAR1) agonist for bleeding in a patient under treatment with an antiplatelet drug.

Brief Description of Drawings

These and other aspects, features and advantages which can be achieved by the present invention will become apparent from and elucidated with reference to the following description of embodiments according to the present invention, in which:

FIG. 1A is a diagrammatic representation of acute Aortic Dissection (AD) showing separation of the inner and middle layers of the aorta, B is a diagrammatic representation of the aorta which relates the degree of dilation to the affected tissue, and C is an open thoracic descending aortic replacement procedure, in which the aorta is divided and sutured into a surgical dacron insertion graft,

FIG. 2 shows the original trajectory illustrating the effect of two stages of human platelet aggregation (upper trajectory) and the secretion of ADP from dense granules (lower trajectory) and autocrine via P2Y12 receptor signaling on the second and irreversible stages of platelet aggregation,

figure 3 shows the original aggregation trajectories in light transmission in plasma of untreated (control) platelets stimulated with collagen and SFLLRN mimicking thrombin, both known physiological platelet agonists, and synthetic sulfated alpha-L-fucoside chain carbohydrate polymers (average monomer unit of 34 fucose molecules (C34)), natural fucoidan or dextran sulfate (DxS) from Fucus Vesiculosus (FV), as well as platelets pretreated with the potent TP alpha receptor antagonist ICI192,605 and the widely administered P2Y12 receptor antagonist AR-C66096.

Fig. 4 shows the degree of aggregation of human platelets in plasma according to light transmission. (A) Experimental results of platelet aggregation in plasma of all administered agonists (as in figure 3) in the presence of AR-C66096 (as a well-known and accepted in vitro P2Y12 receptor antagonist) are shown. (B) Experimental results of platelet aggregation for all administered agonists in the presence of ICI192,506 (a potent TP α receptor antagonist) are shown. (C) Experimental results of platelet aggregation of the above agonists in the presence of both AR-C66096 and ICI192,506 are shown. In terms of light transmission, platelet aggregation was recorded for 20 minutes. Data for the n-5 experiments are expressed as mean ± s.e.m., and statistically analyzed by one-way ANOVA followed by Bonferroni multiple comparison test; for FIGS. 4A-C: p is not more than 01, P is not more than 001, and ns is not significant.

Figure 5 shows the aggregation (in terms of light transmission) of isolated platelets induced by an increase in the concentration of a synthetic sulfated alpha-L-fucoside chain sugar polymer with a monomer chain length of 329 monomers (C329), 34 monomers (C34) and 13 monomers (C13).

Detailed Description

The following description focuses on embodiments of the present invention applicable to treatment strategies of patients receiving antiplatelet drug therapy. Particularly in the treatment of patients suffering from bleeding complications during/after surgery.

During physiologic primary hemostasis, platelets immediately adhere to the bare subendothelial membrane at the site of vascular injury, activate, and release autocrine mediators such as thromboxane a2(TXA2) and Adenosine Diphosphate (ADP), causing the conformation of the fibrinogen receptor integrin α IIb β 3 to change to an active state and eventually aggregate to form a fibrinogen-bridged plug. For TXA2, the thromboxane/prostanoid receptor- α (TP α) is the major isoform of platelets and, like the receptors for thrombin (PAR-1 and PAR-4), is coupled to G α 12/13 and G α q. For ADP, the G α q-coupled P2Y1 receptor and the G α i 2-coupled P2Y12 receptor have been identified on platelets. It is known that signaling through the β/γ subunit of G α i is associated with amplification of granule secretion and activation of integrin α IIa β 3 (termed 'inside-out' signaling), while the α subunit down-regulates Adenylate Cyclase (AC) levels and thus cyclic adenosine monophosphate (cAMP) levels, all of which are critical for adequate and sustained platelet activation and aggregation.

Human platelet aggregation induced by physiological agonists (as shown in figure 2) is characterized by two stages. The first phase is induced by the primary agonist itself and is reversible due to inactivation of the fibrinogen receptor integrin α IIa β 3 if the stimulus is too weak to trigger release of an autocrine feedback mediator (such as ADP) from the compact particle. When the primary stimulus is strong enough to induce ADP secretion, ADP amplifies and enhances platelet response, including activation of integrin α IIa β 3, by autocrine action of P2Y12 receptor signaling, leading to a second and irreversible stage of platelet aggregation.

Although primary platelet agonists induce platelet activation and aggregation to varying degrees depending on intermediate TXA2 generation and autocrine signaling, to date all known physiological platelet agonists, regardless of their initial signaling cascade, are critically dependent on ADP release and subsequent P2Y12 signaling. The combined inhibition of TXA2/TP α signaling by aspirin, for example, and ADP/P2Y12 signaling by ticagrelor, for example, represents the "gold standard" for current antithrombotic therapies. Most, if not all, patients who experience AMI or stroke are under this dual antiplatelet therapy regimen (DAPT) to prevent recurrence of the thromboembolic event.

Thus, for patients under anti-platelet therapy, a first phase of platelet aggregation may occur, but as shown in fig. 2, this first phase is reversible. The effect is shown in figure 3, where dual platelet inhibition (blocking of TP α/TXA2 and P2Y12/ADP receptors) allows only the first but reversible phase of platelet aggregation, which is responsive to collagen and the hexapeptide SFLLRN, which mimics thrombin and PAR-1 activation.

Platelet aggregation is prevented or reversed in patients receiving antiplatelet drugs. When these patients require immediate or acute surgery, bleeding complications of varying degrees are inevitable.

Fucoidans, i.e. polysulfated fucose polysaccharides with different chain lengths from natural sources, have hitherto been proposed for use in clinical settings to have procoagulant or anticoagulant properties, depending on reported or stated observations. However, the precise mode of action at the molecular level has been elusive. In WO 2008103234, it is suggested that purified fucoidan can be used as an anticoagulant for patients with pre-thrombotic conditions such as deep vein thrombosis, arterial thrombosis and other cardiovascular diseases.

The role of fucoidan in platelet activation and aggregation has not been exploited. It has been previously reported that native fucoidan isolated from Fucus vesiculosus induces human and murine platelet activation and aggregation through C-type lectin-like receptor 2 (Clec-2). In studies related to the present disclosure, these findings were not reproducible and platelet endothelial receptor 1(PEAR1) was instead identified as the primary receptor that elicits human platelet activation and aggregation by natural fucoidans as well as synthetic sulfated alpha-L-fucoside chain glycopolymers [1 ]. Prior to the confirmation of the involvement of PEAR-1, synthetic sulfated alpha-L-fucoside chain glycopolymers have been shown to be potent agonists of human platelet activation and aggregation [2,3 ]. However, it has been reported that PEAR1 is also activated by dextran sulfate; the latter was therefore included in studies related to the present disclosure as a positive control for PEAR 1-induced signaling. In Kauskot et al [4], the PEAR1 extracellular domain protein (abbreviated as PEAR1-EC) was shown to induce aggregation of washed (isolated) human platelets in a concentration-dependent manner, however, the possible clinical application of PEAR1-EC was not indicated.

Along this route, the endogenous ligands of PEAR1 in humans and other species remain to be elucidated.

With respect to the two stages of platelet aggregation described above and shown in fig. 2, it was observed that natural fucoidan (from fucus) and synthetic sulfated alpha-L-fucoside chain sugar polymers (chain length 34 monomers (C34)) induced a "classical" two-stage process of platelet aggregation in plasma. A similar trace was observed when platelets were stimulated with dextran sulfate (fig. 3).

In addition, in the presence of receptor antagonists interfering with TXA2/TP α and ADP/P2Y12 signaling (dual inhibition), various degrees of primary and reversible platelet aggregation by collagen and the hexapeptide SFLLRN mimicking PAR-1 activation by thrombin have been observed, and, as expected, lack of secondary and irreversible stages.

In sharp contrast and most notably, as disclosed herein, it was shown that natural fucoidan (from fucus), synthetic sulfated alpha-L-fucoside chain sugar polymer (chain length 34 monomers (C34)), and dextran sulfate trigger the first stage of platelet aggregation, which to our surprise was still irreversible and persistent in the presence of both TP alpha and P2Y12 antagonists (dual inhibition; DAPT).

The data are summarized in fig. 4, which shows that P2Y12 receptor signaling (fig. 4A), TP α receptor signaling (fig. 4B), or both (fig. 4C) are prevented to varying degrees, but that human platelet aggregation in plasma persisting as induced by collagen or by the PAR-1 agonist SFLLRN, which mimics thrombin, is significantly and significantly prevented. In contrast, although single or double inhibition also significantly prevented second-wave platelet aggregation in response to synthetic sulfated alpha-L-fucoside chain sugar polymers (chain length 34 monomers (C34)), native fucans (from fucus) and dextran sulfate, the first-wave platelet aggregation induced by the latter three compounds remained irreversible and sustained for the duration of the assessment (20 minutes) and was significantly higher in extent than that induced by collagen or SFLLRN (except for dextran sulfate, which can be explained by a relatively small number of repetitions (n ═ 5)).

In other words, it was found that agonists of the PEAR1 receptor appear to be capable of inducing approximately half-maximal and sustained aggregation of human platelets in plasma despite blocking P2Y12 receptor signaling, TP α receptor signaling, or both P2Y12 and TP α receptor signaling (dual inhibition; dual antiplatelet therapy; DAPT).

Thus, it was found that the use of platelet endothelial aggregation receptor 1(PEAR1) agonists (herein, fucosan, synthetic sulfated alpha-L-fucoside chain glycopolymers, and dextran sulfate) can result in approximately half maximal and sustained platelet aggregation for patients receiving mono-or bi-antiplatelet drugs. Importantly, platelet endothelial aggregation receptor 1(PEAR1) agonist treatment will thus be directly associated with reducing and minimizing bleeding complications for patients receiving antiplatelet therapy.

In one embodiment, a platelet endothelial aggregation receptor 1(PEAR1) agonist is used to treat bleeding in a patient, wherein the patient is under antiplatelet drug therapy.

Thus, platelet endothelial aggregation receptor 1(PEAR1) agonists provide a viable therapeutic option to address bleeding complications in a large number of patients under single or dual antiplatelet therapy regimens.

Such treatments are typically administered at the bleeding site for local action, such as by local administration of a platelet endothelial aggregation receptor 1(PEAR1) agonist at the wound or bleeding site.

In one embodiment, the administration is topical administration.

In one embodiment, the administration is topical administration at the wound or bleeding site.

For larger lesions, assessment of the extent of the lesion is preferred to provide information for optimal lesion treatment. In cases of severe bleeding, administration of a platelet endothelial aggregation receptor 1(PEAR1) agonist may be combined with other hemostatic measures, such as suturing or temporary use of a tourniquet.

There are several different antiplatelet drugs such as acetylsalicylic acid (aspirin), non-steroidal anti-inflammatory drugs (NSAIDs), dipyridamole, P2Y12 Adenosine Diphosphate (ADP) receptor antagonists, and integrin α IIb β 3 function inhibitors. The combined inhibition of TXA2/TP α signaling by aspirin, for example, and ADP/P2Y12 signaling by ticagrelor, for example, is standard antithrombotic therapy.

In one embodiment, the antiplatelet agent or antiplatelet therapy comprises one or more agents that inhibit TXA2 synthesis and/or act as TP α receptor antagonists and/or one or more agents that act as ADP receptor inhibitors and/or act as P2Y12 receptor antagonists.

In one embodiment, the antiplatelet agent or antiplatelet therapy comprises one or more agents selected from the group consisting of acetylsalicylic acid, triflusal (triflusal) and terutroban and/or one or more agents selected from the group consisting of ticlopidine (ticlopidine), clopidogrel, cangrelor, prasugrel (prasugrel), eligrel (elinogrel) and ticagrelor.

The patient population under such drug treatment is broad and includes stroke patients, patients with heart disease, and patients who have undergone surgery.

In one embodiment, the patient has or has had a condition selected from the group consisting of: stroke with or without atrial fibrillation, cardiac surgery, prosthetic replacement of heart valves, coronary heart disease (such as stable angina, unstable angina and heart attack), patients with coronary stents, peripheral vascular disease/peripheral arterial disease and apex/ventricle/mural thrombosis, coronary artery disease, heart attack, stents or coronary artery bypass graft surgery (CABG).

Antiplatelet therapy is becoming more and more effective and modern antiplatelet therapies can maintain their effectiveness for as long as 72 hours after administration. While this is a clear advantage in terms of convenience for patients under life-long treatment, it represents a significant risk if the patient requires an unplanned immediate surgery.

It is crucial to be able to stop bleeding during or after a patient is under surgery for antiplatelet therapy, and in particular when the patient has received a high prophylactic dose of antiplatelet agent, such as within 72, 48, 24, 12, such as 6, 5, 4, 3, 2 or 1 hour. Such bleeding complications include patients who receive continuous single or double anti-platelet drugs and require immediate surgery or dental procedures.

In one embodiment, the bleeding is acute bleeding.

In one embodiment, the bleeding is caused by surgery.

In one embodiment, the antiplatelet agent or antiplatelet therapy has been administered to the patient within 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hours.

One specific example of such a patient population is associated with acute Aortic Dissection (AD), which is a catastrophic manifestation of aortic intimal tear. Blood enters the false lumen from the true lumen by tearing, causing the inner and middle layers of the aorta to separate (dissect) as can be seen in fig. 1A and 1B.

The deeper the dissection, the deeper the ballooning developed and the more tissue was adversely affected by perfusion (fig. 1B). This blood supply deficiency actually explains several symptoms similar to AMI or stroke.

A swedish population-based study of 14.229 individuals (including high necropsy rates) showed an incidence of AD of 3.4 cases/100.000 persons/year. Including the estimated number of unreported cases, the International Registry of Acute Aortic dissection (IRAD) evaluated the incidence of AD per 100.000 persons (16 men and 7.9 women), respectively, with a mean age of 63 years. In contrast, in this age group, the incidence of Acute Myocardial Infarction (AMI) experienced per year in 100.000 residents in sweden is about 400.

In this regard, the symptoms of AD are largely similar to those of severe thrombotic events, such as AMI or stroke, and can include, for example, sudden severe chest or upper back pain, shortness of breath, sudden speech difficulties, loss of vision, or weakness or paralysis of one side of the body. Because of these similarities and morbidity probabilities, when patients present with one or more of the above symptoms and are sent to emergency hospitals, they are immediately administered a high prophylactic dose of an antiplatelet drug, such as clopidogrel, cangrelor or ticagrelor, which targets the platelet P2Y12/ADP receptor. In one embodiment, the antiplatelet agent or antiplatelet therapy comprises administration of the agents clopidogrel, cangrelor and/or ticagrelor.

In one embodiment, the antiplatelet agent or antiplatelet therapy is dual antiplatelet therapy (DAPT). Such a therapy may be 60-90mg ticagrelor, twice daily, in combination with 70-100mg aspirin.

Immediate surgery is inevitable when a patient is subsequently diagnosed as experiencing AD, rather than AMI or stroke. Typically, this requires an open thoracic aortic replacement procedure in which the aorta is divided and sutured into a surgical dacron insertion graft. Surgical access is through a large incision in the left side of the chest.

At the junction between the aorta and the graft, as well as at the suture, bleeding is prolonged due to the lack of platelet aggregation, which is a direct result of initial receipt of an ultra-high dose of a P2Y12 receptor antagonist (such as clopidogrel, cangrelor, or ticagrelor).

Until bleeding stops, the patient may need to infuse up to 30 blood bags and/or open the chest for up to two days in an intensive care unit. In economic terms, every minute of each transfusion and opening of the chest greatly increases the risk of serious infection, increasing the already very critical condition of the patient.

Thus, for acute Aortic Dissection (AD) patients undergoing immediate surgery after receiving ultra-high doses of anti-P2Y 12 platelet drugs, bleeding complications can be managed and stopped using platelet endothelial aggregation receptor 1(PEAR1) agonists.

In one embodiment, the patient has acute Aortic Dissection (AD).

In one embodiment, the patient has received a high dose of a P2Y12 receptor antagonist, such as 180-480mg ticagrelor.

In one embodiment, the patient is initially misdiagnosed as experiencing AMI or stroke and has therefore received a high dose of a P2Y12 receptor antagonist, such as 180-.

In one embodiment, the platelet endothelial aggregation receptor 1(PEAR1) agonist is a polysulfated polysaccharide and/or sulfated saccharide polymer. As demonstrated by the data in this application, all of these agonists elicit platelet aggregation.

FIG. 3 shows sustained platelet aggregation of sulfated carbohydrate polymers (sulfated alpha-L-fucoside chain carbohydrate polymers), native fucoidan from Fucus vesiculosus, and dextran sulfate; the received data are summarized in fig. 4. For all of these platelet endothelial aggregation receptor 1(PEAR1) agonists, irreversible first wave platelet aggregation is clearly seen, although TP α/TXA2 signaling, P2Y12/ADP signaling is inhibited and when both pathways are blocked (dual antiplatelet therapy; DAPT).

The chemical composition of natural fucoidan is quite different due to the ecological physiological parameters. The first structure was elucidated in 1950 from fucoidan extracted from Fucus vesiculosus. It was subsequently determined that fucoidan consists of 50-90% L-fucose, 35-45% sulphate esters and less than 8% uronic acids, with a linear backbone of α (1 → 2) -glycosidic linkages based on O-4 sulfated L-fucose and some sugars such as galactose, mannose, xylose and glucose. A revised structure of commercial fucoidan from Fucus vesiculosus was later published, indicating that it consists mainly of an alpha (1 → 3) -L-fucose linear backbone, with sulfate substitutions at O-4 and some alpha-L-fucose branching at O-4 or O-2.

In one embodiment, the polysulfated polysaccharide is a fucan and/or dextran sulphate.

In one embodiment, the polysulfated polysaccharide is a fucan.

In one embodiment, the fucoidan is extracted from brown algae or brown seaweed.

In one embodiment, the fucoidan is extracted from fucus.

However, the heterostructure of natural fucoidan may pose problems in such applications. Furthermore, the degree of sulfation may vary from purification batch to purification batch, which may be directly related to its effect. It is difficult to obtain a completely pure sample after purification, and this potential batch diversity complicates administration.

Thus, it is recognized that synthetic fucose-mimicking glycopolymers (such as sulfated alpha-L-fucoside side chain glycopolymers) may exhibit several advantages.

In one embodiment, the polysulfated saccharide polymer is a sulfated alpha-L-fucoside chain saccharide polymer.

The fucoidan-mimicking carbohydrate polymers can be synthesized by several different means, such as by cyanato-mediated free radical polymerization, thiol-mediated chain transfer free radical polymerization techniques, or RAFT polymerization. In the present invention, RAFT polymerisation was found to provide unbranched polysaccharide chains and to provide good control of polymer chain length.

In one embodiment, the sulfated alpha-L-fucoside chain sugar polymer comprises an unbranched chain of side chain carbohydrates.

In the present invention, artificially synthesized sulfated fucose polymers (sulfated carbohydrate polymers that mimic fucoidans) of non-human and non-animal origin are compared with purified fucoidans. It was found that the synthetic sulfated carbohydrate polymers caused the same sustained effect on platelet aggregation as was observed for the purified fucoidan (fig. 3, fig. 4).

The chain length is selected to provide sufficient biological activity at the applied concentration while still being short enough to ensure the synthesis of a homogeneous fraction. In addition, the chain length appears to be suitable for synthesizing carbohydrate polymers or coupling carbohydrate polymers to linker/adapter proteins.

As shown in fig. 5, isolated platelets were induced by sulfated carbohydrate polymers of several different chain lengths of mock fucan. Here, chain lengths of 13, 34 and 329 monomers (monosaccharide units) all trigger complete and sustained aggregation of isolated platelets (see also [3 ]). The dose-response curves shown clearly show that the shorter the monomer chain length of the synthetic sulfated fucose polymer, the higher the concentration required to cause the same degree of platelet aggregation.

In one embodiment, the unbranched chain of the side chain carbohydrate contains at least 10 monosaccharide units, such as at least 13 monosaccharide units.

In one embodiment, the unbranched chain of the side chain carbohydrate contains from 10 to 500 monosaccharide units, such as from 10 to 350 monosaccharide units, such as from 13 to 329 monosaccharide units.

In one embodiment, the unbranched chain of the side chain carbohydrate contains 13, 34 or 329 monosaccharide units.

The artificially synthesized sulfated saccharide polymer is sulfated alpha-L-fucoside chain poly (methacrylamide). The final product is exemplified in formula 1 below:

in one embodiment, the sulfated saccharide polymer has the general formula 1, which isWherein R is SO₃And (4) Na. In a further embodiment, n is 10 to 500, such as 13 to 329, such as 13, 34 or 329.

In one embodiment, the sulfated saccharide polymer is a sulfated alpha-L-fucoside chain poly (methacrylamide).

To provide sulfated carbohydrate polymers that mimic fucoidan, all carbohydrate polymers were O-sulfated with sulfate esters. In the present invention, high levels of sulfation are preferred, such as more than 50% sulfation, such as more than 60%, more than 70%, more than 80% or more than 90% sulfation.

In one embodiment, the fucan or sulfated saccharide polymer is sulfated to an extent of at least 50%, such as at least 60%, preferably at least 75%, such as at least 85%. In one embodiment, the fucan or sulfated saccharide polymer is sulfated to an extent of 50% to 100%, such as 75% to 95%, such as 85% to 90%.

The synthesized sulfated saccharide polymers have several advantages. They can be highly purified and the length as well as the degree of sulfation can be controlled. The sulfated levels of the synthesized saccharide polymers all exceeded 75%, and for the synthesized saccharide polymer in fig. 5, the sulfated level of C34 was as high as 87%, the sulfated level of C13 was as high as 89%, and the sulfated level of C329 was as high as 87%. The side chain carbohydrate chains also simplify the attachment of the synthetic fucose-mimetic carbohydrate polymers and provide a uniform filling or layer of attached fucose-mimetic carbohydrate polymers.

The approximate average molecular weight of the sulfated saccharide polymers is below 500000g/mol, such as below 300000 g/mol. In one embodiment, the approximate average molecular weight of the sulfated sugar polymer is 3500 to 275000g/mol, such as 7000 to 180000 g/mol.

The sulfated carbohydrate polymers of the synthetic simulated fucans can have incorporated chemical functional groups, such as linkers for linking the carbohydrate polymers of the synthetic simulated fucans to a surface or substrate. These features may be biotinylation for streptavidin affinity binding, pegylation to increase solubility, prolong stability and reduce immunogenicity or chemical cross-linking, etc.

Dextran is a complex branched polyglucose (a polysaccharide derived from the condensation of glucose). IUPAC defines glucans as "microbial-derived branched poly-alpha-d-glucosides with glycosidic linkages predominantly C-1 → C-6". The glucan chains have different lengths (3 to 2000 kilodaltons). Dextran sulfate is a dextran containing about 13% to 21% (such as 17%) sulfur (17% corresponds to about 2.3 sulfate groups/glucosyl residues). Dextran sulfate is a heparin analog.

In one embodiment, the polysulfated polysaccharide is dextran sulfate.

In one embodiment, the dextran sulfate has an approximate average molecular weight of 250000g/mol or more, such as 250000 to 2000000g/mol, such as 500000g/mol or more, such as 500000 to 2000000 g/mol.

In one embodiment, the approximate sulfur content of dextran sulfate is 13% to 21%, such as 15% to 19%, such as 17%.

In one embodiment, the approximate sulfur content of dextran sulfate is about 1.8 to 2.8, such as 2.0 to 2.6, such as 2.3 sulfate groups/glucosyl residues.

The platelet endothelial aggregation receptor 1(PEAR1) agonist in the present invention may be in any suitable form, including a powder, a gel, a solution, or any combination of these. To facilitate the use of platelet endothelial aggregation receptor 1(PEAR1) agonists, it may also be in the form of a topical hemostatic, sealant or adhesive agent for use during surgery. In the specific example of acute Aortic Dissection (AD), such powders, gels, solutions or topical hemostats, sealants or adhesives may be used at the junction between the aorta and the graft as well as at the sutures.

Topical hemostatic agents, sealants and adhesives are normally used in the art, i.e., until the desired effect is achieved. However, in the present invention, these may be used for patients under treatment with antiplatelet drugs.

In one embodiment, there is provided a local hemostatic, sealant or adhesive, wherein the local hemostatic, sealant or adhesive comprises a platelet endothelial aggregation receptor 1(PEAR1) agonist according to any one of claims 1 to 18 for use in treating bleeding in a patient, wherein the patient is under antiplatelet drug therapy.

In one embodiment, the topical hemostatic agent, sealant or adhesive is used in treating bleeding in a patient, wherein the use is topical.

In one embodiment, the topical hemostatic agent, sealant or adhesive is used in treating bleeding in a patient, wherein the use is treating bleeding in a patient during surgery.

In one embodiment, a medical patch comprising a topical hemostatic agent, sealant or adhesive is used to treat bleeding in a patient, wherein the patient is under antiplatelet drug therapy.

In one embodiment, the local hemostatic, sealant or adhesive is for local administration during surgery, wherein the local hemostatic, sealant or adhesive comprises a platelet endothelial aggregation receptor 1(PEAR1) agonist for use in treating bleeding in a patient under treatment with an antiplatelet drug. In one embodiment, the platelet endothelial aggregation receptor 1(PEAR1) agonist is a sulfated alpha-L-fucoside chain glycopolymer, a native fucan, or a dextran sulfate for use in treating bleeding in a patient under treatment with an antiplatelet agent. In a further embodiment, the medical patch comprises a topical hemostatic agent, sealant, or adhesive.

Materials and methods

Chemical product

Collagen was obtained from Chrono-Log (Chrono-Log Corporation, Havertown, PA, USA) and the PAR1 activating Peptide SFLLRN was custom synthesized by JPT Peptide Technologies (berlin, germany). Fucoidan (95% pure) from Fucus vesiculosus was purchased from Sigma Aldrich Sweden AB (stockholm, Sweden). Synthetic sulfated alpha-L-fucoside chain sugar polymers having an average monomer unit of 13, 34 or 329 fucose molecules are the university of forest snow (C.)

University, forest snow Flat, Sweden) physical, chemical and biological (IFM)/chemical (KEMI) line Peter Konrassson professor friendly gift and was generated as described previously [2,3]. The ADP/P2Y12 receptor antagonist AR-C66096 and the TXA2/TP α receptor antagonist ICI192,605 were purchased from Tocris, UK.

Preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP)

Warp beam

The county area ethics committee (Dnr 2015/543) approved that blood was obtained by venipuncture from healthy volunteers who denied taking any medication two weeks prior to donation, and inhaled into a 10ml syringe (Sarstedt, N ü mbrecht, germany, order No.: 02.1067.001) pre-filled with trisodium citrate solution by the supplier. Blood was centrifuged at 220g for 20 minutes. The PRP thus obtained was transferred to a fresh 15ml polypropylene tube, and the remaining blood was centrifuged at 2200g for 20 minutes to separate PPP from the remaining blood cells.

Platelet aggregation in PRP

Measurements were performed at 37 ℃ using a Chrono-Log luminescence aggregometer (model 700, Chrono-Log Corporation, Havertown, PA, USA) with a final volume of 0.3ml of platelet suspension stirred at 900 revolutions per minute. Agglutination is expressed as percent light transmission compared to PPP alone (═ 100%).

Platelets were preincubated with vehicle or AR-C66096 for 5 minutes as indicated, followed by stimulation with 1. mu.g/ml collagen, 10. mu.M SFLLRN, 30. mu.g/ml C34, 10. mu.g/ml fucoidan, or 30. mu.g/ml dextran sulfate (DxS) (FIG. 4A).

Platelets were preincubated with vehicle or ICI192,506 for 5 minutes as indicated, followed by stimulation with 1. mu.g/ml collagen, 10. mu.M SFLLRN, 30. mu.g/ml C34, 10. mu.g/ml fucoidan, or 30. mu.g/ml dextran sulfate (DxS) (FIG. 4B).

Platelets were preincubated with vehicle or AR-C66096 and ICI192,506 for 5 minutes as indicated, followed by stimulation with 1. mu.g/ml collagen, 10. mu.M SFLLRN, 30. mu.g/ml C34, 10. mu.g/ml fucoidan, or 30. mu.g/ml dextran sulfate (DxS) (FIG. 4C).

Sugar polymer synthesis

2-methacrylamidoethyl 2,3, 4-tri-O-acetyl- α -L-fucopyranoside was dissolved in 1, 4-dioxane (1.20M) and CPBDT was added. The solution was transferred to a tube containing AIBN and a stir bar and treated with N₂(g) Rinsing for 30 minutes. The tube was sealed and placed in an oil bath preheated to 90 ℃. The solution was stirred at 90 ℃ for 24 hours, diluted with 1, 4-dioxane, and freeze-dried. The crude solid compound was purified by suspension in ether, centrifuged, and isolated. This process was repeated a total of three times to yield a peracetylated dithiobenzoate capped sugar polymer as a pink powder. The peracetylated dithiobenzoate capped sugar polymer was then dissolved in 1, 4-dioxane (0.45mL/mmol monomer units). The solution was transferred to a tube with a magnetic stirrer and AIBN (6.6 eq/sugar polymer chain). Will the pipe N₂(g) Rinsing for 30 minutes. The tube was sealed and immersed in an oil bath preheated to 90 ℃. The solution was stirred overnight, diluted with 1, 4-dioxane, and lyophilized. The crude peracetylated sugar polymer was suspended in ether, centrifuged, and separated to yield the peracetylated sugar polymer as a white powder. This process was performed a total of three times. The peracetylated saccharide polymer was suspended in methanol (0.08mL/mmol monomer unit), sodium methoxide (1 eq/mol monomer unit) was added, and stirred at room temperature overnight. The solution was neutralized with Dowex Marathon C (H +), filtered, and the solvent was evaporated. The crude sugar polymer was dissolved in deionized water and purified by dialysis against deionized water for 48 hours, with the deionized water being changed frequently. Lyophilization afforded the sugar polymer as a white loose powder, i.e., the sugar polymer.

To provide a fucoidan-mimicking glycopolymer, polymer (2) is sulfated by the following reaction: the sugar polymer was dissolved in DMF (0.01mL/mmol monomer units) with vigorous stirring. Sulfur trioxide-pyridine complex (5 equivalents/free hydroxyl group) was added and the solution was stirred at room temperature for 44 hours. The solution was decanted and the precipitated crude O-sulfated saccharide polymer was dissolved in 0.9M NaCl (aq) (90mmol NaCl/mmol monomer units). The solution was stirred at room temperature for 46 hours and dialyzed against 0.1M NaCl for 19 hours and then against deionized water for 24 hours, with the deionized water being changed frequently. The resulting solution was lyophilized to give the O-sulfated saccharide polymer as a white loose powder.

The synthesis yields sulfated polymers with sulfation degrees ranging from 87 to 89%. To calculate the effect of adding sulfate, the approximate average molecular weight of C13 would be about 7260g/mol, the approximate average molecular weight of C34 would be about 16470g/mol, and the approximate average molecular weight of C239 would be about 178300 g/mol.

Results

In the results shown in fig. 4A, the effect of interfering with P2Y12 receptor signaling on human platelet aggregation in plasma induced by collagen, PAR-1 activating peptide SFLLRN, synthetic sulfated α -L-fucoside chain sugar polymer (average monomer unit of 34 fucose molecules (C34)), natural fucoidan from Fucus Vesiculosus (FV), and dextran sulfate (DxS) was evaluated. AR-C66096 is used as a well-known and accepted in vitro P2Y12 receptor antagonist (a precursor ultimately developed into AR-C66931 MX), which is more broadly known as cangrelor, or trade name

As expected, the extent of platelet aggregation induced by collagen and SFLLRN was effectively reduced, from 85 ± 2% to 23 ± 4% and from 91 ± 3% to 5 ± 1%, respectively. Reactions induced by synthetic sulfated alpha-L-fucoside chain glycopolymers (C34: 92 + -3% down to 53 + -6%, P.ltoreq.0.001), native fucoidan from Fucus Vesiculosus (FV) (96 + -2% down to 43 + -4%, P.ltoreq.0.001), and dextran sulfate (DxS) (84 + -5% down to 23 + -6%, P.ltoreq.0.001) were also significantly and significantly affected by AR-C66096. However, except for experiments with dextran sulfate, the extent of these latter reactions remains significantly higher in all settings than those elicited by collagen or SFLLRN in the presence of AR-C66096.

As shown in fig. 4B, we next studied and evaluated the effect of interfering with TXA2 release and subsequent TP α receptor signaling on human platelet aggregation in plasma induced by collagen, PAR-1 activating peptide SFLLRN, synthetic sulfated α -L-fucoside chain glycopolymer (average monomer unit of 34 fucose molecules (C34)), native fucoidan from Fucus Vesiculosus (FV), and dextran sulfate (DxS). Instead of using acetylsalicylic acid (ASA; aspirin), we used ICI192,605, a potent antagonist of the TP α receptor, to prevent intercellular TXA2 synthesis.

Platelet aggregation induced by collagen and SFLLRN was significantly reduced, from 90 + -3% to 21 + -4% and from 91 + -3% to 15 + -6%, respectively. Reactions induced by synthetic sulfated alpha-L-fucoside chain glycopolymers (C34: 91 + -3% down to 45 + -7%, P.ltoreq.0.001), native fucoidan from Fucus Vesiculosus (FV) (96 + -2% down to 44 + -4%, P.ltoreq.0.001), and dextran sulfate (DxS) (88 + -6% down to 39 + -7%, P.ltoreq.0.001) were affected to a lesser extent but still significantly by ICI192,605. Nevertheless, except for experiments with dextran sulfate, the extent of these latter reactions was still significantly higher in our setting than those elicited by collagen or SFLLRN in the presence of ICI 165,605.

We finally evaluated human platelet aggregation in plasma triggered by collagen, PAR-1 activating peptide SFLLRN, synthetic sulfated alpha-L-fucoside chain glycopolymer (average monomer unit of 34 fucose molecules (C34)), natural fucoidan from Fucus Vesiculosus (FV) and dextran sulfate (DxS) under conditions in which both P2Y12 receptor and TP alpha receptor are blocked by AR-C66096 and ICI192,605, respectively. Platelets were preincubated with vehicle or AR-C66096 and ICI192,506 for 5 minutes as indicated, followed by stimulation with 1. mu.g/ml collagen, 10. mu.M SFLLRN, 30. mu.g/ml C34, 10. mu.g/ml fucoidan from Fucus Vesiculosus (FV), or 30. mu.g/ml dextran sulfate (DxS). The results are summarized in fig. 4C. Platelet aggregation by collagen and SFLLRN was significantly inhibited from 86 + -2% to 11 + -2% and from 93 + -2% to 3 + -1%, respectively. Reactions induced by synthetic sulfated alpha-L-fucoside glycopolymers (C34: 92 + -3% down to 48 + -9%, P.ltoreq.0.001), native fucoidan from Fucus Vesiculosus (FV) (95 + -2% down to 44 + -6%, P.ltoreq.0.001), and dextran sulfate (DxS) (88 + -6% down to 37 + -6%, P.ltoreq.0.001) were less prominent but still significantly reduced by the dual platelet inhibition of AR-C66096 and ICI192,605. Nevertheless, the extent of these latter reactions, except for some using dextran sulfate, is still significantly higher in our setting than those elicited by collagen or SFLLRN in the presence of AR-C66096 and ICI192,605.

Although the present invention has been described above with reference to one or more specific embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and, other embodiments than the specific above are equally possible within the scope of these appended claims (e.g., different from those described above).

In the claims, the term "comprising" does not exclude the presence of other elements or steps. Furthermore, although individually listed, a plurality of means, elements or method steps may be implemented by e.g. a single unit or processor. Furthermore, although individual features may be included in different claims, these may possibly be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms "a", "an", "first", "second", etc do not exclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

Reference documents:

[1]C.Kardeby,K.Falker,E.J.Haining,M.Criel,M.Lindkvist,R.Barroso,P.Pahlsson,L.U.Ljungberg,M.Tengdelius,G.E.Rainger,S.Watson,J.A.Eble,M.F.Hoylaerts,J.Emsley,P.Konradsson,S.P.Watson,Y.Sun,M.Grenegard,Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbalpha,Blood Adv 3(3)(2019)275-287,10.1182/bloodadvances.2018024950.

[2]M.Tengdelius,C.J.Lee,M.Grenegard,M.Griffith,P.Pahlsson,P.Konradsson,Synthesis and biological evaluation of fucoidan-mimetic glycopolymers through cyanoxyl-mediated free-radical polymerization,Biomacromolecules 15(7)(2014)2359-68,10.1021/bm5002312.

[3]M.Tengdelius,C.Kardeby,K.Falker,M.Griffith,P.Pahlsson,P.Konradsson,M.Grenegard,Fucoidan-Mimetic Glycopolymers as Tools for Studying Molecular and Cellular Responses in Human Blood Platelets,Macromol Biosci 17(2)(2017),10.1002/mabi.201600257.

[4]Alexandre Kauskot,Michela Di Michele,Serena Loyen,Kathleen Freson,Peter Verhamme,and Marc F.Hoylaerts,Anovel mechanism of sustained platelet IIbβ3activation via PEAR1,BLOOD,26 April 2012,Volume 119,Number 17.

Claims (22)

1. a platelet endothelial aggregation receptor 1(PEAR1) agonist for use in treating a hemorrhage in a patient, wherein the patient is under antiplatelet drug therapy.

2. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to claim 1, wherein the antiplatelet drug therapy has been administered to the patient within 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hours.

3. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 2, wherein the antiplatelet drug therapy comprises: one or more agents that inhibit TXA2 synthesis and/or act as TP alpha receptor antagonists, and/or

One or more drugs as ADP receptor inhibitors and/or as P2Y12 receptor antagonists.

4. A platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 3, wherein the antiplatelet drug therapy comprises: one or more drugs selected from acetylsalicylic acid, triflusal and terlutripan, and/or

One or more drugs selected from the group consisting of ticlopidine, clopidogrel, cangrelor, prasugrel, eletgrelor and ticagrelor.

5. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 4, wherein the hemorrhage is an acute hemorrhage.

6. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 5, wherein the patient has or has had a condition selected from the group consisting of: stroke with or without atrial fibrillation, cardiac surgery, prosthetic replacement of heart valves, coronary heart disease (such as stable angina, unstable angina and heart attack), patients with coronary stents, peripheral vascular disease/peripheral arterial disease and apex/ventricle/mural thrombosis, coronary artery disease, heart attack, stents or coronary artery bypass graft surgery (CABG).

7. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 6, wherein the patient has acute Aortic Dissection (AD).

8. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 7, wherein the patient was initially misdiagnosed as having experienced an Acute Myocardial Infarction (AMI) or stroke and has therefore received a high dose of a P2Y12 receptor antagonist.

9. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 1 to 8, wherein the platelet endothelial aggregation receptor 1(PEAR1) agonist is a polysulfated polysaccharide and/or a sulfated saccharide polymer.

10. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to claim 9, wherein the polysulfated polysaccharide is a fucan and/or dextran sulphate.

11. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to claim 10, wherein the fucoidan is extracted or purified from brown or brown seaweed.

12. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to claim 9, wherein the sulfated carbohydrate polymer is a sulfated alpha-L-fucoside chain carbohydrate polymer.

13. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to claim 12, wherein the sulfated alpha-L-fucoside chain sugar polymer comprises an unbranched chain of side chain carbohydrates.

14. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to claim 13, wherein the unbranched chain of side chain carbohydrates contains 10 to 500, such as 10 to 350 monosaccharide units.

15. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 9 to 14, wherein the sulfated carbohydrate polymer is a sulfated alpha-L-fucoside chain poly (methacrylamide).

16. Platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 9 to 15, wherein the fucan and/or sulfated alpha-L-fucoside chain glycopolymer is sulfated to an extent of at least 50%, such as at least 60%, preferably at least 75%, such as at least 85%.

17. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 9 to 16, wherein the dextran sulfate has an approximate average molecular weight of 250000g/mol or more.

18. The platelet endothelial aggregation receptor 1(PEAR1) agonist for use according to any one of claims 9 to 17, wherein the dextran sulfate has an approximate sulfur content of 13% to 21%, such as 15% to 19%, such as 17%.

19. A topical hemostatic, sealant or adhesive, wherein the topical hemostatic, sealant or adhesive comprises a platelet endothelial aggregation receptor 1(PEAR1) agonist according to any one of claims 1 to 18 for use in treating bleeding in a patient, wherein the patient is under antiplatelet drug therapy.

20. A topical hemostat, sealant or adhesive according to claim 19 for use in treating bleeding in a patient wherein the use is topical.

21. The topical hemostat, sealant or adhesive according to claim 19 or 20 for use in treating bleeding in a patient, wherein the use is treating bleeding in a patient during surgery.

22. A medical patch comprising the topical hemostatic, sealant or adhesive of any one of claims 19 to 21 for use in treating a hemorrhage in a patient, wherein the patient is under antiplatelet drug therapy.

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